The present invention relates to novel compounds of formula I 
and/or of formula II 
in which R1, R2, R3, R4, R5, X1, X2, X3, X4, X5 and Y are defined as described below. The compounds of formulae I and II inhibit cyclin-dependent kinases (CDKs) and other kinases (e.g. KDR), have cytostatic actions, and are particularly suitable for treating tumors. The compounds of formulae I and II are obtainable by extracting the plant Uvaria klaineri, PLA 100484, by cell cultures of the plant Uvaria klaineri, or by chemical synthesis. Thus, the invention relates to a method for preparing the compounds of formulae I and II, to the use of the compounds for producing a pharmaceutical for the treatment of malignant disorders and of diseases which may be treated by inhibition of CDKs and KDR, and to pharmaceutical compositions comprising at least one compound of the formulae I or II.
Cancer is a disease of humans and animals which is in most cases fatal and which is caused by the uncontrolled growth of endogenous cells. The term xe2x80x9ccancerxe2x80x9d denotes the formation of malignant growths (malignancies) and neoplasms (tumors or carcinomas), or the malignant degeneration and dysmaturity of white blood cells (leukemia). Cancer or tumor cells are formed by transformation of endogenous cells. The malignancy of the cancer cell expresses itself in the autonomy of growth, i.e. in its capability of growing uninhibitedly, without integration into the organ system, and infiltrating, resulting in the destruction of tissue. A sure sign of malignancy is the formation of metastases far from the tumor after hematogenic or lymphogenic spreading of tumor cells. Cancer is one of the most frequent causes of death in humans, and there is therefore a great demand for methods and agents for curing or treating malignant degenerations. In addition to thexe2x80x94if possible radicalxe2x80x94surgical removal of the tumor, the options for therapy of malignant tumors include radiotherapy with X-rays, xcex1-, xcex2-, xcex3-rays, immunotherapy and chemotherapy. As yet, the use of immunotherapy is limited. Chemotherapy of tumors is understood as meaning administration of cell toxins (cytostatics) for the treatment of tumors and remaining tumor cells after local surgical treatment or irradiation. These substances intervene specifically in certain processes of cell division, so that tissues having a high proportion of dividing cells, such as rapidly growing tumor tissue, react more sensitively. The agents used are alkylating compounds, such as cyclophosphamide (The Merck Index, 12th Ed. page 463), antimetabolites, such as methotrexate (The Merck Index, 12th Ed. page 1025), alkaloids, such as vincristine (The Merck Index, 12th Ed. page 1704), antibiotics, such as daunomycin (The Merck Index, 12th Ed. page 479), and adriamycin (The Merck Index, 12th Ed. pages 581-582). However, owing to massive side-effects, all these agents have great disadvantages, so that the death of the diseased individual is only delayed, but not prevented. Furthermore, degenerated (cancer) cells become resistant to the agents used. When this occurs, the conventional pharmaceuticals no longer have any cytostatic action, but they are toxic, owing to the side-effects. Furthermore, it has been found that a combined and/or sequential use of cytostatics exceeds the activity of an individual cytostatic (monotherapy), and it is therefore possible that the considerable side-effects in polychemotherapy are non-additive. For all these reasons, novel chemotherapeutics are urgently required and thus investigated worldwide.
Surprisingly, it has been found that the African plant Uvaria klaineri is capable of forming highly effective novel cytostatics which inhibit cell growth even at very low concentrations. The novel compounds are denoted klainetins below and are, together with klainetin derivatives, the subject of the invention. The klainetins are substituted phenols which inhibit cyclin-dependent kinases and therefore influence cell cycle regulation. Since in cancers cell proliferation is abnormally accelerated and regulation is out of control, CDK inhibitors are valuable agents for the treatment of malignant degenerations. The compounds of formula I belong to the substituted 2,3-dihydroflavone type and the ring structure corresponds to that of isochamanetin which has been described most recently by H. Achenbach et al. in Phytochemistry, vol. 44, pages 359-364 (1997). The compounds of the formula I according to the invention are novel substituted isochamanetins.
The compounds of formula II belong to the chalcone class of compounds; the ring structure and carbon skeleton correspond to that of uvaretin. Uvaretin has been found in many Uvaria species (M. H. H. Nkunya et al., Planta Med. 57:341-343 (1991); I. Muhammad and P. G. Waterman, J. Nat. Prod. 48:571-80 (1985)). The inventive compounds of the formula II are distinguished from uvaretin by substitution and dehydrogenation and have not yet been described in the literature. Owing to their different chemical structure, the compounds of the formulae I and II have novel and physicochemical, biological and pharmacological properties. In contrast, the substances known up until now often have disadvantages which are expressed in an unsatisfactory action level, high toxicity and/or undesired side-effects.
The present invention therefore relates to compounds of formula I 
and/or of formula II 
wherein
R1, R2, R3, R4 and R5 independently of one another are H, C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, unsubstituted or substituted with one or more OH;
X1, X2, X3, X4 and X5 independently of one another are O, NH, Nxe2x80x94C1-C6-alkyl, Nxe2x80x94C2-C6-alkenyl, Nxe2x80x94C2-C6-alkynyl, acyl, aryl or S;
Y is one or more H, halogen, OH, Oxe2x80x94C1-C6-alkyl, Oxe2x80x94C2-C6-alkenyl, Oxe2x80x94C2-C6-alkynyl, NH2, NHxe2x80x94C1-C6-alkyl, NHxe2x80x94C2-C6-alkenyl, NHxe2x80x94C2-C6-alkynyl, NH-acyl, SH, Sxe2x80x94C1-C6-alkyl, Sxe2x80x94C2-C6-alkenyl, Sxe2x80x94C2-C6-alkynyl, acyl or aryl;
wherein the compounds may be in any stereochemical form, mixtures of said forms in any ratio, physiologically tolerated salts, and/or chemical equivalents thereof.
In the formulae I and II
C1-C6-alkyl is a straight-chain or branched alkyl having 1 to 6 carbon atoms such as methyl, ethyl, isopropyl, tert-butyl and hexyl,
C2-C6-alkenyl is a straight-chain or branched alkenyl having 2 to 6 carbon atoms such as allyl, crotyl and pentenyl, and
C2-C6-alkynyl is a straight-chain or branched alkynyl having 2 to 6 carbon atoms such as propynyl, butynyl und pentynyl.
Aryl can be, for example, phenyl, benzyl or 1- or 2-naphthyl, which may also be unsubstituted or substituted, for example, by halogen such as chlorine, bromine and fluorine, by alkyl having 1-4 carbon atoms, preferably methyl, by hydroxyl, by alkoxy having 1-4 carbon atoms, in particular methoxy, and/or by trifluoromethoxy.
Acyl can be aliphatic or aromatic acyl radicals. Aliphatic acyl has 1-7, preferably 1-4, carbon atoms, such as formyl, acetyl, propionyl, butyryl, hexanoyl, acryloyl, crotonoyl, propioloyl, which may be further substituted, for example, by halogen such as chlorine, bromine and fluorine, by amino and/or by alkylamino having 1-4 carbon atoms, preferably methyl- or ethylamino groups. Aromatic acyl can be, for example, benzoyl or naphthoyl, which may also be further substituted, for example, by halogen such as chlorine, bromine and fluorine, by alkyl having 1-4 carbon atoms, preferably methyl, by hydroxyl, by amino groups such as ethylamino, or by alkoxy groups having 1-7, preferably 1-4, carbon atoms, in particular methoxy.
In formula I, X1R1, X2R2 and X3R3 preferably are OH, X4R4 preferably is Oxe2x80x94C1-C4-alkyl and Y is H. In the formula II, X1R1, X2R2, X3R3, and X4R4 preferably are OH, X5R5 preferably is Oxe2x80x94C1-C4-alkyl and Y is H.
The invention thus relates to klainetin A of the formula IA 
in all its stereochemical forms, to mixtures of said forms in any ratio, and to physiologically tolerated salts thereof.
The invention further relates to klainetin B of the formula IIA 
According to the invention, the compounds of the formulae I and II are obtainable by extracting the plant Uvaria klaineri or any of its variants or mutants under suitable conditions. The klainetins are produced by subsequent isolation of the compounds and, where appropriate, conversion into chemical derivatives and physiologically tolerated salts thereof.
The invention therefore relates furthermore to a method for preparing a compound of formulae I and/or II, which comprises culturing the plant Uvaria klaineri or any of its variants or mutants under suitable conditions, until one or more klainetins of formulae IA and/or IIA accumulate in the plant material, are subsequently isolated from the plant parts and, where appropriate, are converted into chemical derivatives and/or physiologically tolerated salts.
The plant Uvaria klaineri, its mutants and/or variants are preferably grown on suitable soil in a tropical or subtropical climate, until the novel klainetins accumulate in the plant. The novel klainetins are subsequently isolated from the plants and, where appropriate, fractionated into the individual active components.
The plants are produced preferably under tropical conditions, for example, at a temperature between 18 and 45xc2x0 C. and high humidity.
The product can also be produced in cell cultures. To this end, living cells of the plant Uvaria klaineri are transferred into a suitable culture medium and the cell cultures are grown until the compounds of the invention accumulate in the medium. Cell cultures are prepared often from callus cultures. The culture media comprises minerals, vitamins, carbon sources such as sucrose, and nitrogen sources such as nitrogen salts. The klainetins are then isolated from the cell culture and, where appropriate, fractionated into the individual active components.
Many reactions for the chemical modification of flavones or phenols are described in the literature. The phenolic OH groups of the present compounds therefore can be derivatized using chemical reactions known in the art. It is, for example, possible to achieve reduction to the saturated compounds catalytically with hydrogen or to reduce carbonyl groups using metal hydrides auch as aluminum hydrides or borohydrides. Another suitable example is the reaction of carbonyl groups with hydroxylamine or its derivatives to give the oximes which can be chemically converted further.
The invention is described in detail below, in its several embodiments.
The klainetins of the invention are produced by Uvaria klaneri. Uvaria klaineri is a widespread evergreen liana of the Annonaceae family. The Annonaceae family includes many tropical useful and medicinal plants. Their geographical range is the entire tropics; the sample from which the klainetins were isolated was collected in Gabon, specifically in the area around Rabi/Gamba.
When isolating klainetins, it is possible to use other species from the genus Uvaria or from plants of the same species which come from a different location. Klainetin content and composition may vary depending on the conditions at the location, such as soil quality, temperature, humidity, and incidence of light.
The method of the invention may be used for extraction and isolation on a laboratory scale (100 g to 1 kg of dry plant material) and on an industrial scale (100 to  greater than 1000 kg).
Uvaria klaineri generates a mixture of klainetins in the plant material. Depending on the harvest of the plant and its parts, the quantitative proportion of one or more of the klainetins of the invention may vary. In addition, it is possible to control the synthesis of individual klainetins via the growth conditions so that one or more of the klainetins fail to be generated or are generated in an amount below the detection limit.
The plant material preferably contains a detectable klainetin. Preference is given to the formation of klainetins A and B.
In addition to klainetins A and B (compounds of the formulae IA and IIA, respectively), the plant Uvaria klaineri also generates further related compounds which are distinguished from the compounds represented by the formulae IA and IIA by altered hydroxylation or glycosylation. For example, a klainetin with a molecular mass of 408 Da was detected as by-product.
The plants may be cultured outdoors or, preferably, in a greenhouse. Alternatively, it is possible to use plant cell cultures for producing the metabolites. Normally, the starting material is callus cultures. Selection of suitable bioreactors for growing the plant cell culture makes it possible to achieve optimal mixing and airing of the culture without disturbing the plant cells, cell growth and metabolite production. The cultures may be mixed using, for example, airlift reactors or bubble column reactors, or blade or propeller stirrers. The cells can grow as individual cells or as branched or unbranched cell aggregates or cell chains. Metabolite production can be induced by stimulation with exogenous factors, for example heavy metal salts or plant elicitors.
Product formation in the plant can be monitored on the basis of the pH of the cultures and by chromatographic methods such as thin layer chromatography, high pressure liquid chromatography, or by testing the biological activity. The klainetins of the invention are contained both in the leaves and in other parts of the plant. The isolation method described below serves to purify the klainetins of the invention, preferably to purify klainetins A and B.
The klainetins of the invention were isolated and purified from the plant or the culture medium according to known methods with respect to the chemical, physical and biological properties of the natural substances. The klainetin concentrations in the starting material or in the individual isolation stages can be assayed by thin layer chromatography, for example, on silica gel with chloroform/methanol/glacial acetic acid/water mixtures (e.g. in a quantitative 8:1:1:0.2 ratio) as the mobile phase, or HPLC. For thin layer chromatography fractionation, detection can be carried out, for example, using color reagents such as I2 vapor, iron(III) chloride, vanilline-H2SO4 or Pauli""s reagent (sulfanilic acid, diazotized), the amount of the substance formed being. compared with a standard solution.
The klainetins of the invention are isolated by harvesting the plant, i.e., by collecting the leaves, stems, wood, bark or roots, and then by extracting the plant, which preferably is still fresh or is dried according to the conventional methods. Next, the klainetins are extracted from the plant material, optionally with a water-contaning organic solvent. The organic solvent phase contains the klainetins of the invention, which are, where appropriate, concentrated in vacuo and further purified, as described below.
The extracts are combined, diluted with water, and extracted with a suitable organic solvent immiscible with water, for example n-butanol. The subsequently removed organic phase is, where appropriate, concentrated in vacuo. Lipids can be removed from the product of interest by diluting the concentrate with a nonpolar solvent in which the klainetins of the invention have very low solubility, such as hexane, petroleum ether, or diethyl ether. Here, the kainetins precipitate, and the lipophilic contaminants such as waxes remain in solution and are removed by conventional solid/liquid phase separations. The precipitate which contains all klainetins is dissolved in water/methanol in 1/30 of the original volume. The precipitate is dissolved in the process and is lyophilized. The lyophilisate, denoted xe2x80x9ccrude productxe2x80x9d below, contains 0.5 to 10% klainetins and is used for further isolation.
One or more of the klainetins of the invention are further purified by chromatography on suitable materials, for example, on molecular sieves, on normal phase supports such as silica gel and aluminum oxide, on ion exchange materials, on adsorber resins, or on reversed phase (RP) materials. The klainetins are separated with the aid of said chromatography. Klainetin chromatography is carried out with buffered aqueous solutions or mixtures of aqueous and organic solutions.
The term xe2x80x9cmixtures of aqueous and organic solutionsxe2x80x9d means all organic solvents miscible with water, for example, methanol, 2-propanol and acetonitrile, at a concentration of from 10 to 80% solvent, preferably 15 to 55%, solvent, or else all buffered aqueous solutions which are miscible with organic solvents. The buffers to be used are the same as those mentioned above.
The klainetins are separated due to their different polarity with the aid of reversed phase chromatography, for example on MCI(copyright) (adsorber resin from Mitsubishi, Japan) or Amberlite XAD(copyright) (TOSOHAAS), on other hydrophobic materials, for example, on RP-8 or RP-18 phases, or else on xe2x80x9cpolyamidesxe2x80x9d. In addition, the separation may be carried out with the aid of normal phase chromatography, for example on silica gel, aluminum oxide and the like.
Klainetin chromatography is carried out with buffered or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other organic solvents miscible with water. The organic solvents usually used are propanol and acetonitrile.
The term xe2x80x9cbuffered or acidified aqueous solutionsxe2x80x9d means, for example, water, phosphate buffer, ammonium acetate, citrate buffer at a concentration of from 1 mM to 0.5 M, and also formic acid, acetic acid, trifluoroacetic acid and all commercially available acids known to the skilled worker, at a concentration of from 0.01 to 3%, generally, 0.1%.
Chromatography is carried out using a gradient which starts with 100% aqueous buffer and ends with 100% solvent; preference is given to running a linear gradient of 10 to 50% 2-propanol or acetonitrile.
Alternatively, it is also possible to carry out gel chromatography or chromatography on hydrophobic phases.
Gel chromatography is carried out on polyacrylamide gels or mixed polymer gels such as Biogel-P 2(copyright) (Biorad), Fractogel TSK HW 40(copyright) (Merck, Germany or Toso Haas, USA), or on Sephadex(copyright) (Pharmacia, Uppsala, Sweden).
The order of the abovementioned chromatographies is reversible.
Another very effective purification step for klainetins is crystallization. The klainetins crystallize from solutions in organic solvents and from mixtures of water with organic solvents. Crystallization is carried out in a manner known in the art, for example by concentrating or cooling saturated klainetin solutions.
The klainetins of the invention are stable in the solid state and in solutions having a pH in the range between 3 and 8, in particular 4 and 6, and can thus be worked into customary pharmaceutical preparations.
The klainetins and derived chemical derivatives of the formulae I and II can be converted into the corresponding physiologically tolerated salts according to methods known to the skilled worker.
The term xe2x80x9cphysiologically tolerated saltsxe2x80x9d of compounds of the formulae I and II means both organic and inorganic salts thereof, as described in Remington""s Pharmaceutical Sciences (17th edition, page 1418 (1985)). Owing to their physical and chemical stability and to solubility, sodium, potassium, calcium and ammonium salts, are inter alia preferred for acidic groups; salts of hydrochloric acid, sulfuric acid, phosphoric acid or of carboxylic acids or sulfonic acids, such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid, are inter alia preferred for basic groups.
The invention furthermore includes obvious chemical equivalents of the compounds of the formulae I and II, which are slightly different chemically, i.e. they have the same effectiveness or can be converted into the compound of the invention under mild conditions. Said equivalents include, for example, esters and ethers and also reduction products of the compounds of the invention.
Ester and ether derivatives and reduction products can be prepared according to methods described in the literature, for example in Advanced Organic Synthesis, 4th edition, J. March, John Wiley and Sons., 1992.
The present invention includes all stereoisomeric forms of the compounds of the formulae I and II. Asymmetry centers contained in the compounds of the formulae IA and IIA may all have, independently of one another, either S configuration or R configuration. The invention includes all possible enantiomers and diastereoisomers and likewise mixtures of two or more stereoisomeric forms, for example mixtures of enantiomers and/or diastereoisomers, at all ratios. The invention thus relates to enantiomers in enantiomerically pure form, both as levorotatory and dextrorotatory antipodes, R and S configurations, in the form of racemates and in the form of mixtures of the two enantiomers at all ratios. If cis/trans isomerism is present, the invention relates to both the cis and the trans form and to mixtures of these forms at all ratios. Assays for determining biological klainetin activities:
The assay systems comprise a kinase of the CDK family, CDK4 (cyclin-dependent kinases), and KDR (tyrosine kinase receptor), which play an important part in cell cycle regulation and angiogenesis. Through phosphorylation reactions, CDK4 initiates a reaction cascade at the end of which the cell cycle progresses from the G1 phase to the S phase. Thus, further cell division and further unregulated cell growth can be stopped if this key enzyme is inhibited. KDR is a tyrosine kinase receptor which plays a key part in endothelium growth and angiogenesis and is likewise involved in tumor formation. Thus, CDK4 and KDR represent important therapeutic target molecules for cancers and other proliferative disorders. The assay involves measuring CDK4 and KDR kinase activities on the basis of phosphorylation of a specific peptide substrate. In addition to the inhibiting activity on the kinases mentioned, other kinases which are involved in the formation of cancer and in the inflammation cascade were also inhibited by the klainetins.
Owing to their valuable pharmacological properties, the compounds of the invention are suitable for specific application as pharmaceuticals in human and/or veterinary medicine. The compounds of the invention may be used for cancers, in particular as chemotherapeutics. Due to their cytostatic properties, in particular their strong antitumor activity, and also to an antimicrobial action, they may be used in particular as cytostatics for malignant degenerations in animals and humans.
In tumor cells which have developed resistances against conventional agents, only novel agents have a therapeutically sufficient action. Thus, the klainetins of the invention and chemical derivatives thereof of formulae I and II potentially have an outstanding action even against these problem cell types.
The invention also relates to pharmaceutical preparations which contain one or more of the klainetins of the invention and/or chemical derivatives thereof. Preference is given to their use in a mixture with suitable excipients or carrier material. Carrier materials which may be used in humans are all pharmacologically acceptable carrier materials and/or excipients.
The invention also relates to a method for preparing a pharmaceutical of the invention which comprises combining at least one of the compounds of the invention with a pharmaceutically suitable and physiologically tolerated carrier and, where appropriate, with further suitable active substances, additives or excipients, into a form that can be administerated suitably.
The pharmaceuticals of the invention are generally administered orally, locally or parenterally, but rectal administration is in principle also possible. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, capsules, microcapsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in ampoules and/or preparations with protracted active substance release. Production of these preparations usually involves using carriers and additives and/or aids such as disintegrants, binders, coating agents, swelling agents, glidants or lubricants, flavorings, sweeteners or solubilizers. Frequently used carriers or excipients include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactalbumin, gelatin, starch, vitamins, cellulose and its derivatives, animal or vegetable oils, polyethylene glycols and solvents such as sterile water, alcohols, glycerol and polyhydric alcohols.
It is possible, where appropriate, to microencapsulate the dosage units for oral administration in order to delay the release or to prolong it over a longer period, for example by coating or by embedding the active substance in particle from in suitable polymers, waxes or the like.
Preference is given to producing and administering the pharmaceutical preparations in dosage units, each unit containing as the active ingredient a particular dose of one or more compounds of the klainetins of the invention and/or chemical derivatives thereof. For solid dosage units such as tablets, capsules and suppositories, said dose may be up to about 500 mg, but is usually about 0.1 to 200 mg, and for injection solutions in ampoules up to about 500 mg, but usually about 0.1 to 100 mg, per day.
The daily dose to be administered depends on the body weight, age, sex and state of the patient. However, higher or lower daily doses may also be appropriate under certain circumstances. The daily dose may be administered both by a single administration in the form of a single dosage unit or else in several smaller dosage units and by multiple administrations of subdivided doses in particular intervals.